We would like to compare the RPE1 with the K562 essential Perturb-seq experiments by computing the correlations between Z-scaled dWUI values across experiments. First, we must compute comparable WUI values using the same isoforms as were used in the K562 essential data.
We can also use this to compare the baseline (non-targeting pertubations).
library(magrittr)
library(tidyverse)
library(ggbeeswarm)
library(Matrix)
library(matrixStats)
library(SummarizedExperiment)
library(pheatmap)
library(caret)
set.seed(20210818)
## filtering options
## number of cells in perturbation
MIN_CELLS=30
## max NTPs with NA WUI
MAX_NTP_NA=0.20
## min avg TPM in NTPs
MIN_MEAN_TPM=20
## with few replicates, we need a prior on minimum sd(WUI)
## this avoids div by zero when computing Z-dWUI
## when all baseline replicates have identical counts
MIN_SD_WUI=0.01
## inputs
FILE_SE="data/se/rd7_essential_bulk_expressed.Rds"
FILE_UTRS="../gwps/tbl/df_utrs_kd6_essential_ui10.csv"
FILE_GENES="tbl/df_gene_nnclusters_kd6_essential_ui10.csv"
GENE_CLUSTERS_NONRESPONSE=c("9","12","13")
FILE_CLUSTERS="tbl/df_target_nnclusters_kd6_essential_ui10.csv"
FILE_CLUSTER_MAP="metadata/kd6-cluster-info-sm.csv"
## output files
date_str <- format(Sys.Date(), "%Y%m%d")
FILE_WUI_RPE1=sprintf("data/dwui/%s-wui-target-gene-rd7-essential-kd6-matched.Rds", date_str)
FILE_DWUI_RPE1=sprintf("data/dwui/%s-dwui-target-gene-rd7-essential-kd6-matched.Rds", date_str)
FILE_ZDWUI_RPE1=sprintf("data/dwui/%s-zdwui-target-gene-rd7-essential-kd6-matched.Rds", date_str)
## visuals
NCOLORS=100
COLORS_BWR <- colorRampPalette(c("blue", "white", "red"))(NCOLORS)
BREAKS_DWUI <- seq(-0.8, 0.8, length.out=NCOLORS + 1)
BREAKS_ZDWUI <- seq(-6, 6, length.out=NCOLORS + 1)
BREAKS_ZDWUI_BROAD <- seq(-10, 10, length.out=NCOLORS + 1)
df_utrs <- read_csv(FILE_UTRS)
df_genes <- read_csv(FILE_GENES, col_types='ccc') %>%
mutate(is_nonresponse=cluster_id %in% GENE_CLUSTERS_NONRESPONSE)
df_clusters <- read_csv(FILE_CLUSTERS, col_types='cccc')
se_tx_target <- readRDS(FILE_SE)
## consider only genes used in Perturb-seq clustering
idx_genes <- df_genes %>% filter(!is_nonresponse) %$% unique(gene_id)
idx_txs <- df_utrs %>%
filter(gene_id %in% idx_genes,
transcript_id %in% rownames(se_tx_target)) %$%
unique(transcript_id)
se_tx_target %<>% `[`(idx_txs, .$n_cells >= MIN_CELLS)
## attach UTR annotations from K562 Perturb-seq
rowData(se_tx_target) %<>% as_tibble %>%
left_join(df_utrs, by=c("transcript_id", "transcript_name",
"gene_id", "gene_name", "utr_rank")) %>%
mutate(seqnames=NULL, end=NULL, strand=NULL) %>%
DataFrame(row.names=.$transcript_id) %>%
`[`(idx_txs,)
idx_ntp_rd7 <- colnames(se_tx_target) %>% { .[str_detect(., "non-targeting")] }
idx_target_rd7 <- colnames(se_tx_target) %>% { .[. %in% df_clusters$sgID_AB] }
se_tx_target %<>% `[`(, c(idx_ntp_rd7, idx_target_rd7))
## cluster annotations
id2cluster <- c(
setNames(rep_along(idx_ntp_rd7, "ntp"), idx_ntp_rd7),
deframe(df_clusters[,c("sgID_AB", "cluster_id")])
)
df_annots <- enframe(id2cluster, name='rownames', value='cluster_id') %>%
column_to_rownames('rownames') %>%
as.data.frame
cts_tx_target <- assay(se_tx_target, "counts")
M_gene_tx <- rowData(se_tx_target) %>% as_tibble %>%
dplyr::select(transcript_id, gene_id) %>%
deframe %>%
fac2sparse
cts_gene_target <- M_gene_tx %*% cts_tx_target
ui_tx_target <- cts_tx_target / (t(M_gene_tx) %*% cts_gene_target)
wt_tx <- rowData(se_tx_target) %>% as_tibble %>%
dplyr::select(transcript_id, utr_wt) %>% deframe
wui_gene_target <- M_gene_tx %*% Diagonal(length(wt_tx), wt_tx) %*% ui_tx_target
## identify genes that are sufficiently expressed in the
## NTPs to compute WUIs
idx_genes <- rowMeans(is.na(wui_gene_target[,idx_ntp_rd7])) %>%
{ names(.)[. <= MAX_NTP_NA] }
wui_gene_target %<>% `[`(idx_genes,)
awui_ntp <- rowMeans(wui_gene_target[,idx_ntp_rd7], na.rm=TRUE)
dwui_gene_target <- apply(wui_gene_target, 2, function (x) x - awui_ntp)
dwui_gene_target[is.na(dwui_gene_target)] <- 0
pheatmap(t(dwui_gene_target[,idx_target_rd7]),
show_colnames=FALSE, show_rownames=FALSE,
scale='none', treeheight_row=0, treeheight_col=0,
color=COLORS_BWR, breaks=BREAKS_DWUI, annotation_row=df_annots)
pheatmap(t(dwui_gene_target[,idx_target_rd7]),
show_colnames=FALSE, show_rownames=FALSE,
scale='column', treeheight_row=0, treeheight_col=0,
color=COLORS_BWR, breaks=BREAKS_ZDWUI, annotation_row=df_annots)
swui_ntp <- rowSds(as.matrix(wui_gene_target[, idx_ntp_rd7]), na.rm=TRUE)
## set minimum stdev
swui_ntp[swui_ntp < MIN_SD_WUI] <- MIN_SD_WUI
zdwui_gene_target <- apply(dwui_gene_target, 2, function (x) x/swui_ntp)
pheatmap(t(zdwui_gene_target[,idx_target_rd7]),
show_colnames=FALSE, show_rownames=FALSE,
treeheight_col=0, treeheight_row=0,
scale='none', annotation_row=df_annots,
color=COLORS_BWR, breaks=BREAKS_ZDWUI)
pheatmap(t(zdwui_gene_target[,idx_target_rd7]),
show_colnames=FALSE, show_rownames=FALSE,
treeheight_col=0, treeheight_row=0,
scale='none', annotation_row=df_annots,
color=COLORS_BWR, breaks=BREAKS_ZDWUI_BROAD)
saveRDS(wui_gene_target, file=FILE_WUI_RPE1)
saveRDS(dwui_gene_target, file=FILE_DWUI_RPE1)
saveRDS(zdwui_gene_target, file=FILE_ZDWUI_RPE1)
We can coherently compute Z-dWUIs for 869 genes in 646 targets that correspond with the targets that had clustering in the K562 essential screen. The baseline is estimated with 112 non-targeting perturbations.
## R version 4.2.2 (2022-10-31)
## Platform: x86_64-apple-darwin13.4.0 (64-bit)
## Running under: macOS Big Sur ... 10.16
##
## Matrix products: default
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##
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## attached base packages:
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prefix: /Users/mfansler/miniconda3/envs/bioc_3_16